Chitosan‐Gelatin‐EGCG Nanoparticle‐Meditated LncRNA TMEM44‐AS1 Silencing to Activate the P53 Signaling Pathway for the Synergistic Reversal of 5‐FU Resistance in Gastric Cancer

Abstract Chemoresistance is one of the leading causes of therapeutic failure in gastric cancer (GC) treatment. Recent studies have shown lncRNAs play pivotal roles in regulating GC chemoresistance. Nanocarriers delivery of small interfering RNAs (siRNAs) to silence cancer‐related genes has become a novel approach to cancer treatment research. However, finding target genes and developing nanosystems capable of selectively delivering siRNAs for disease treatment remains a challenge. In this study, a novel lncRNA TMEM44‐AS1 that is related to 5‐FU resistance is identified. TMEM44‐AS1 has the ability to bind to and sponge miR‐2355‐5p, resulting in the upregulated PPP1R13L expression and P53 pathway inhibition. Next, a new nanocarrier called chitosan‐gelatin‐EGCG (CGE) is developed, which has a higher gene silencing efficiency than lipo2000, to aid in the delivery of a si‐TMEM44‐AS1 can efficiently silence TMEM44‐AS1 expression to synergistically reverse 5‐FU resistance in GC, leading to a markedly enhanced 5‐FU therapeutic effect in a xenograft mouse model of GC. These findings indicate that TMEM44‐AS1 may estimate 5‐FU therapy outcome among GC cases, and that systemic si‐TMEM44‐AS1 delivery combined with 5‐FU therapy is significant in the treatment of patients with recurrent GC.

.  or  xenografted nude mice were treated with vehicle or 5-FU (10 mg/kg) (n = 5 per group). Tumors were serially passaged in vivo. Tumor volumes are shown as mean ±SD. Figure S2. After the addition of 5 µg/ml 5-FU into medium, the proliferative ability of sensitive and 5-FU-resistant cells was analyzed through CCK-8 assay. Relative proliferation rates at diverse time periods were calculated compared with that at 0 h. Figure S3. 5-FU treatment could induce up-regulation of TMEM44-AS1 in sensitive cell lines. Figure S4. (A)The expression level of TMEM44-AS1 in GC cell lines and normal epithelial cells, measured by RT-qPCR. (B) The Cancer Genome Atlas (TCGA) database showed TMEM44-AS1 high expression is correlated with poor DFS. (C) TMEM44-AS1 up-regulation was common in different tumor types. Figure S5. Addition of 5 µg/ml 5-FU into medium and the proliferative ability of HGC-27/R and MKN-45/R cells subject to TMEM44-AS1 siRNAs or si-NC (negative control) transfection detected through CCK-8 assay. Proliferation rates at diverse time periods were calculated relative to that at 0 h. Figure S6. Protein-coding genes co-expressed with TMEM44-AS1 as determined by KEGG pathway analysis. Figure S7. Visualizing genes in P53 pathway in our sequencing results. Figure S8. Cell proliferation ability of HGC-27/S and MKN-45/S transfected with Lv-TMEM44-AS1 or Lv-NC was detected by EdU assay. Figure S9. (A) RT-qPCR was used to detect the expression of miR-2355-5p in 5-FU sensitive and resistant cell lines. (B) Comparing differences in the expression levels of miR-2355-5p between GC tumor tissues from recurrent(n=38) and nonrecurrent(n=74) patients. Figure S10. RT-qPCR was used to detect the expression of TMEM44-AS1 after miR-2355-5p level changes.  Figure S13. (A) RT-qPCR and WB was used to detect the expression of PPP1R13L after infection with si-NC, or si-TMEM44-AS1. (B) RT-qPCR and WB was used to detect the expression of PPP1R13L after infection with pre-NC, pre-miR-2355-5p, anti-NC,or anti-miR-2355-5p.(C) RT-qPCR and WB was used to detect the expression of PPP1R13L after infection with si-NC + pre-NC, si-NC + pre-miR-2355-5p, si-TMEM44-AS1+pre-NC, si-TMEM44-AS1+pre-miR-2355-5p, si-NC+anti-NC, si-NC+anti-miR-2355-5p, si-TMEM44-AS1+anti-NC, or si-TMEM44-AS1+anti-miR-2355-5p. Figure S14. The size of the chitosan-gelatin-EGCG (CGE) nanoparticles. Figure S15. CCK8 was used to assess the cytotoxicity of CGE after these CGE were transfected into cells for 48 h. Cell viability was calculated as the percentage of viable cells relative to untreated control cells.

Gene silencing or overexpression
Cells were seeded in 6-well plates and cultured overnight until they had attached to the plates. For gene silencing, after replacing the medium with fresh medium, the siRNAs were transfected into cells using Lipo2000 (Invitrogen) or CGE. ShRNAs were transfected using polybrene (Merck Millipore, Germany). After incubation for 8 h, the cells were washed with PBS, fresh medium was added, and the cells were incubated for another 48 h.
For gene overexpression, cells were seeded into 6-well plates and cultured overnight until they had attached to the plates. Overexpression plasmids were transfected into cells using Lipo3000 (Invitrogen), while lentiviral vectors for overexpression analysis were transfected into cells using polybrene. After incubation for 8 h, the cells were washed with PBS, fresh medium was added, followed by incubation for an additional 48 h. RT-qPCR was used to verify gene silencing or overexpression.

Rapid amplification of cDNA ends (RACE)
To determine the transcription initiation and termination sites for lncRNA TMEM44-AS1, 5'-and 3'-RACE assays were performed using Superscript IV Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific) following the manufacturer's instructions. The sequences of the PCR-amplified 5′-and 3′-end fragments were confirmed by direct sequencing.

Fluorescence in situ hybridization (FISH)
Cy3-labeled TMEM44-AS1 and 18S probes were synthesized by GenePharma (Suzhou, China). A Fluorescent in Situ Hybridization Kit (GenePharma) was used to hybridize the probes to cells. Images were captured on an ultra-highresolution laser confocal microscope (ZEISS LSM880 with Airyscan, Germany).

Nuclear and cytoplasmic separation
Nuclear and cytoplasmic fractions of cells were separated using a nucleus and cytoplasm separation kit Bestbio,Shanghai,China). Approximately 5-10×10 6 cells were centrifuged for 2-3 min (4 °C, 500 × g) and then washed twice using cold PBS. Extract liquid A (200 μl) was added to the cell precipitate, the mixture was shaken on ice for 30 min, and then centrifugated (4 °C, 1,200 × g) for 5 min. The resulting supernatant was considered the cytoplasmic fraction and the precipitate the nuclear fraction.

Dual-luciferase reporter assays
Potential miR-2355-5p binding sites in TMEM44-AS1 and the PPP1R13L 3′-UTR were predicted using starBase and TargetScan (http://www.targetscan.org/). Wild-type and mutant reporter vectors were constructed by Genechem (pmirGLO, Shanghai, China). HEK-293T cells were seeded into 96-well plates, incubated overnight, and then transfected with luciferase reporter vectors using Lipo3000. Relative luciferase activities were measured using a Dual-Luciferase Reporter Assay Kit (Promega, USA) on a Varioskan LUX machine (Thermo Scientific) after 48 h of incubation. Firefly luciferase activity was normalized to that of Renilla (pRL-TK) luciferase and foldchanges in luciferase values calculated.

RIP assays
RIP assays were performed using the Merck Millipore) following the manufacturer's instructions. Simply, cells were lysed with RIP lysis buffer. Then, A/G magnetic beads were used for immunoprecipitation of the antibody and the RNA binding protein. The magnetic beads were immobilized with a magnet and the unbound material was washed away. The RNA was subsequently collected and quantified by RT-qPCR. Input control and normal anti-IgG antibody control were also tested to ensure the accuracy of the detected signals from the protein-bound RNA.

Characterization of the CGE nanoparticle
The samples for characterization were prepared by diluting 1:500 (w/v) with deionized water. The particle size, polydispersity index (PDI), and zeta potential of the CGE nanoparticles were measured using a NanoSizer NanoZS analyzer (Malvern Instruments Ltd., Malvern, UK). All measurements were performed in triplicate. The morphological properties of the CGE nanoparticles were determined using a Tecnal-10 Transmission Electron Microscope (Philips, Netherlands) at an accelerating voltage of 200 kV. Samples were prepared by spreading CGE nanoparticles onto a carbon-coated microscope grid and drying at room temperature. Fourier-transform infrared spectra (FTIR) of EGCG, chitosan, gelatin, CGE nanoparticles, a mixture of EGCG, chitosan and gelatin were measured using a Spectrum GX spectrophotometer (PerkinElmer Inc., MA, USA) in the range of 400-4,000 cm −1 . EGCG concentration in the CGE nanoparticles was determined by measuring the free EGCG content in the dialysis medium using HPLC. Free EGCG was regarded as unloaded EGCG.

In vitro transfection and CGE/FAM-siRNA distribution
Cells were seeded on cover glasses (24 mm × 24 mm; CITOTEST) placed at the bottom of wells in 6-well plates and cultured overnight until they had attached to the plates. The next day, 100 pmol FAM-siRNAs was added to the CGE followed by incubation for 1 h at room temperature. This mixture was then added to cells in the 6well plate and incubated for another 6 h. Nuclei were counterstained with Hoechst 33342. The distribution of CGE/FAM-siRNA was observed by ultra-high resolution laser confocal microscopy (ZEISS LSM880 with Airyscan).

Lysosomal escape of siRNA
Cells were seeded in confocal dishes, incubated overnight, and then transfected with CGE/FAM-siRNAs. After incubating for 1, 3, or 6 h, LysoTracker Red (Beyotime) was diluted with culture medium (1:15,000) and used to mark lysosomes for another 30 min. Hoechst 33342 was used for nuclear staining. The distribution of CGE/FAM-siRNAs and lysosomes was observed using an ultra-high-resolution laser confocal microscope (ZEISS LSM880 with Airyscan).